The Enhanced DNA Replication Fidelity of a Mutant Herpes Simplex Virus Type 1 DNA Polymerase Is Mediated by an Improved Nucleotide Selectivity and Reduced Mismatch Extension Ability

ABSTRACT We previously demonstrated that a recombinant herpes simplex virus containing a mutation within the finger domain of DNA polymerase replicated DNA with increased fidelity. In this study, we demonstrate that, compared with wild-type polymerase, the mutant enzyme exhibited improved nucleotide selectivity and a reduced ability to extend from mismatched primer termini, which would contribute to the increased DNA replication fidelity.

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Sequences at the C-terminus of the herpes simplex virus type 1 UL30 protein are dispensable for DNA polymerase activity but not for viral origin-dependent DNA replication

Nucleic Acids Research ◽

10.1093/nar/21.1.87 ◽

1993 ◽

Vol 21 (1) ◽

pp. 87-92 ◽

Cited By ~ 27

Author(s):

Nigel D. Stow

Keyword(s):

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Dna Polymerase ◽

Herpes Simplex Virus Type ◽

Polymerase Activity ◽

Viral Origin ◽

C Terminus ◽

Simplex Virus

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Restoration of Wild-type Pathogenicity to an Attenuated DNA Polymerase Mutant of Herpes Simplex Virus Type 1

Journal of General Virology ◽

10.1099/0022-1317-67-11-2501 ◽

1986 ◽

Vol 67 (11) ◽

pp. 2501-2506 ◽

Cited By ~ 13

Author(s):

B. A. Larder ◽

J. J. Lisle ◽

G. Darby

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Polymerase ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Wild Type ◽

Simplex Virus

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A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity

Journal of Virology ◽

10.1128/jvi.78.2.650-657.2004 ◽

2004 ◽

Vol 78 (2) ◽

pp. 650-657 ◽

Cited By ~ 25

Author(s):

Ying T. Hwang ◽

Harmon J. Zuccola ◽

Qiaosheng Lu ◽

Charles B. C. Hwang

Keyword(s):

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Dna Polymerase ◽

Herpes Simplex Virus Type ◽

Recombinant Viruses ◽

Conserved Regions ◽

Conserved Region ◽

Simplex Virus

ABSTRACT Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.

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Live Covisualization of Competing Adeno-Associated Virus and Herpes Simplex Virus Type 1 DNA Replication: Molecular Mechanisms of Interaction

Journal of Virology ◽

10.1128/jvi.02476-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4732-4743 ◽

Cited By ~ 21

Author(s):

Daniel L. Glauser ◽

Regina Strasser ◽

Andrea S. Laimbacher ◽

Okay Saydam ◽

Nathalie Clément ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Herpes Simplex Virus Type ◽

Wild Type ◽

Adeno Associated Virus ◽

Early Protein ◽

Simplex Virus ◽

Hsv 1

ABSTRACT We performed live cell visualization assays to directly assess the interaction between competing adeno-associated virus (AAV) and herpes simplex virus type 1 (HSV-1) DNA replication. Our studies reveal the formation of separate AAV and HSV-1 replication compartments and the inhibition of HSV-1 replication compartment formation in the presence of AAV. AAV Rep is recruited into AAV replication compartments but not into those of HSV-1, while the single-stranded DNA-binding protein HSV-1 ICP8 is recruited into both AAV and HSV-1 replication compartments, although with differential staining patterns. Slot blot analysis of coinfected cells revealed a dose-dependent inhibition of HSV-1 DNA replication by wild-type AAV but not by rep-negative recombinant AAV. Consistent with this, Western blot analysis indicated that wild-type AAV affects the levels of the HSV-1 immediate-early protein ICP4 and the early protein ICP8 only modestly but strongly inhibits the accumulation of the late proteins VP16 and gC. Furthermore, we demonstrate that the presence of Rep in the absence of AAV DNA replication is sufficient for the inhibition of HSV-1. In particular, Rep68/78 proteins severely inhibit the formation of mature HSV-1 replication compartments and lead to the accumulation of ICP8 at sites of cellular DNA synthesis, a phenomenon previously observed in the presence of viral polymerase inhibitors. Taken together, our results suggest that AAV and HSV-1 replicate in separate compartments and that AAV Rep inhibits HSV-1 at the level of DNA replication.

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